CN113310958B - Preparation method of a hierarchical porous metal-organic framework chiral sensing probe and the resulting probe and its application - Google Patents

Abstract

The invention relates to a preparation method of a hierarchical porous metal organic framework chiral sensing probe, which comprises the steps of providing a hierarchical porous metal organic framework microsphere with a regular pore channel structure; fixing amino acid oxidase on the hierarchical porous metal organic framework microsphere to obtain the hierarchical porous metal organic framework microsphere with the enzyme fixed, wherein the amino acid oxidase is loaded in a pore channel of the hierarchical porous metal organic framework microsphere through adsorption; and (3) fixing the fluorescent molecules on the hierarchical porous metal organic framework microspheres fixed with the enzyme to obtain the hierarchical porous metal organic framework chiral sensing probe, wherein the fluorescent molecules are loaded in the pore channels of the hierarchical porous metal organic framework microspheres through adsorption. The invention also provides a hierarchical porous metal organic framework chiral sensing probe obtained by the preparation method and application thereof. The invention can identify and analyze the content of amino acid with high sensitivity and high specificity by fixing different amino acid oxidases as chiral identification centers.

Description

A preparation method of a hierarchical porous metal-organic framework chiral sensing probe and thereby The resulting probes and their applications

technical field

The invention relates to enzyme immobilization and analytical chemistry, more specifically to a preparation method of a hierarchical porous metal-organic framework chiral sensing probe, the resulting probe and its application.

Background technique

Amino acids are one of the most dominant and important chiral compounds in nature (Angew. Chem. Int. Ed. 2017, 56, 7276-7281). L-amino acids and D-amino acids are not mutually exclusive and usually coexist as a non-racemic mixture. In mammals and humans, amino acids usually exist in free form or in the form of protein. In general, the majority of amino acids in organisms and nature exist in the L form, while a large amount of the D form usually indicates negative symptoms, aging or disease (ACS Appl. Mater. Interfaces 2017, 9, 20991-20999). The production and racemization of amino acids are of great value in life sciences (Amino Acids 2012, 42, 1553-1582). Amino acid enantiomers are also widely used as chiral sources of asymmetry. The total amount of amino acids in the central nervous system, as well as the ratio of enantiomers usually exhibit different biological functions, and play a crucial role in human physiology and pathology. The expression level of specific chiral amino acids in biological systems is usually Associated with the early stages of many diseases, for example, chronic kidney disease, Alzheimer's disease and cancer (J. Am. Chem. Soc. 2016, 138, 12099-12111). Therefore, rapid and accurate chiral analysis of amino acids is of great significance. So far, there are many methods for the selective identification and detection of amino acid enantiomers, including chromatography, capillary electrophoresis, fluorescence, circular dichroism, etc. (Biosens. Bioelectron. 2020, 151, 111971). However, although conventional chromatography and capillary electrophoresis methods for enantiomer recognition and separation are effective for the chiral detection of amino acids, the cost of the test is high, the procedures and steps required for the test are cumbersome, and the detection time is long. Fluorescence sensors can avoid these shortcomings, and can detect chiral amino acids quickly, effectively, simply and conveniently. This method is suitable for high-throughput screening and real-time imaging of amino acid enantiomers in biological samples. However, the design of chiral binding/reactive sites is the key and great challenge for the enantioselective recognition of fluorescent probes.

To achieve the stereochemical interaction between the substrate and the probe molecule requires a complex and precise chemical synthesis process. At the same time, fluorescent probes are rarely capable of simultaneous enantioselective and chemoselective recognition of specific amino acids, and may be interfered by other similar biochemical molecules. Therefore, it is of great significance to develop a general fluorescent sensing strategy suitable for broad-spectrum detection of chiral amino acids, whether enantioselective or chemoselective.

Contents of the invention

In order to realize the enantioselective and chemoselective recognition of amino acids by fluorescent probes, the present invention provides a preparation method of hierarchical porous metal-organic framework chiral sensing probes, the obtained probes and applications thereof.

According to the preparation method of the hierarchical porous metal organic framework chiral sensing probe of the present invention, it comprises the steps: S1, providing the hierarchical porous metal organic framework microsphere (HPUiO-66), which has a regular pore structure; S2, the amino acid Oxidase (AAO) was immobilized on hierarchical porous metal-organic framework microspheres (HPUiO-66) to obtain enzyme-immobilized hierarchical porous metal-organic framework microspheres (AAO@HPUiO-66), in which amino acid oxidase (AAO) passed Adsorbed and loaded in the pores of hierarchical porous metal-organic framework microspheres (HPUiO-66); S3, immobilized fluorescent molecules (PF) on hierarchical porous metal-organic framework microspheres immobilized with enzymes (AAO@HPUiO-66), and obtained Hierarchical porous metal-organic framework chiral sensing probe (AAO&PF@HPUiO-66), in which fluorescent molecules (PF) are loaded into the pores of hierarchical porous metal-organic framework microspheres (HPUiO-66) by adsorption.

Preferably, the amino acid oxidase (AAO) is an amino acid oxidase with chiral recognition function. More preferably, the amino acid oxidase (AAO) is at least one selected from the group consisting of L-amino acid oxidase, L-glutamic acid oxidase and L-tryptophan oxidase.

Preferably, the fluorescent molecule (PF) is a fluorescent molecule that responds to H ₂ O ₂ . More preferably, the fluorescent molecule (PF) is 3-Oxo-3',6'-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) -3H-spiro[isobenzofuran-1,9'-ton]-6-carboxylic acid.

的Hofmeister盐溶介导作用，合成出具有9nm左右大孔径的分级多孔金属有机骨架微球(HPUiO-66)，合成步骤简单、制备周期短。Preferably, the step S1 includes: S11, using the block copolymer PEO ₁₀₆ PPO ₇₀ PEO ₁₀₆ (F127) to provide a metal organic framework (Metal organic Framework, MOFs) precursor solution for the template; S12, generating a graded by solvothermal Porous metal-organic framework precursor (HPUiO-66-as); S13, Hierarchical porous metal-organic framework microspheres (HPUiO-66) with large pore size obtained by activation and detemplating. More preferably, the pore size of the hierarchically porous metal-organic framework microspheres (HPUiO-66) is 8-10 nm. In a preferred embodiment, the pore size of the hierarchically porous metal organic framework microspheres (HPUiO-66) is 9.2 nm. More preferably, stir and dissolve the block copolymer F127 in deionized water, add sodium perchlorate and acetic acid, and ultrasonically dissolve; then add cerium ammonium nitrate and terephthalic acid, stir and disperse; stir and react at 60°C for 20 minutes; Hierarchical porous metal-organic framework microspheres (HPUiO-66) were obtained after centrifugal separation, washing, activation and drying. Specifically, 150 mg of F127 was dissolved in 9 mL of deionized water, and then 750 mg of NaClO ₄ ·H ₂ O and 450 μL of acetic acid were ultrasonically dispersed in the above F127 solution; subsequently, 249 mg of terephthalic acid and 274 mg of cerium nitrate Ammonium was stirred and dispersed in the above solution; then, the mixed liquid was stirred and reacted at 60°C for 20 minutes; the mixture was centrifuged to obtain a solid product; then the material was washed with deionized water, N,N-dimethylformamide, and absolute ethanol , and soak the material in absolute ethanol at 60°C for 48 hours, during which the ethanol is constantly replaced to remove all the F127 in the pores of the material. After washing and drying, hierarchical porous metal-organic framework microspheres with a size of 600nm-1000nm are finally obtained (HPUiO-66). The present invention utilizes block copolymer F127 as template, utilizes

Hofmeister salt-mediated effect, synthesized hierarchical porous metal-organic framework microspheres (HPUiO-66) with a large pore size of about 9 nm, the synthesis steps are simple and the preparation cycle is short.

Preferably, the step S2 includes: dissolving amino acid oxidase (AAO) with chiral catalytic function in deionized water, adding hierarchical porous metal-organic framework microspheres (HPUiO-66), stirring at 25°C for 4h, and Hierarchical porous metal-organic framework microspheres (AAO@HPUiO-66) immobilized with enzymes were collected by centrifugation.

Preferably, the step S3 includes: dissolving fluorescent molecules (PF) in deionized water, adding hierarchically porous metal-organic framework microspheres (AAO@HPUiO-66) immobilized with enzymes, stirring at 25°C for 30 minutes in the dark, And the hierarchical porous metal-organic framework chiral sensing probe (AAO&PF@HPUiO-66) was collected by centrifugation.

In the present invention, amino acid oxidase (AAO) is a natural enzyme, fluorescent molecule (PF) is a fluorescent small molecule, and the two are sequentially immobilized on the intermediary of hierarchical porous metal-organic framework microspheres (HPUiO-66) through a mild afterloading method. In the pore channel, a series of fluorescent probes with high specificity sensing performance were prepared. Specifically, amino acid oxidase (AAO) and fluorescent molecules (PF) were sequentially immobilized in the hierarchical mesopores of hierarchical porous metal-organic framework microspheres (HPUiO-66) by post-adsorption at room temperature, and a hierarchically porous Metal-organic framework chiral sensing probe (AAO&PF@HPUiO-66), amino acid oxidase (AAO) loaded at 151 mg g ^-1 , fluorescent molecule (PF) immobilized at 374 mg g ^-1 , prepared under mild conditions , Easy to operate.

The present invention also provides a hierarchical porous metal-organic framework chiral sensing probe (AAO&PF@HPUiO-66) obtained by the above preparation method, wherein amino acid oxidase (AAO) and fluorescent molecule (PF) are coupled in the hierarchical porous Metal-organic framework microspheres (HPUiO-66) on the support.

The present invention further provides an application of the above-mentioned hierarchical porous metal-organic framework chiral sensing probe (AAO&PF@HPUiO-66), which has a chiral sensing response to amino acids. Accordingly, the hierarchical porous metal-organic framework chiral sensing probe (AAO&PF@HPUiO-66) was used for the recognition of chiral amino acids through the cascade response system constructed by amino acid oxidase (AAO) and fluorescent molecules (PF).

Preferably, the amino acid is phenylalanine, leucine, methionine, tryptophan, histidine, isoleucine, tyrosine, and/or cysteine.

Preferably, the hierarchical porous metal-organic framework chiral sensing probe (AAO&PF@HPUiO-66) showed fluorescence enhancement for the L-enantiomer of amino acids, but no response for the D-enantiomer of amino acids.

Preferably, the hierarchical porous metal-organic framework chiral sensing probe (AAO&PF@HPUiO-66) is dispersed in HEPES buffer to form a probe solution, the amino acid solution is added to the probe solution for incubation, and the fluorescence intensity is measured by a fluorescence spectrometer.

Preferably, amino acids are linearly analyzed using hierarchical porous metal-organic framework chiral sensing probes (AAO&PF@HPUiO-66) within the concentration range of 0-100 μM in the amino acid solution. Accordingly, the hierarchical porous metal-organic framework chiral sensing probe (AAO&PF@HPUiO-66) can be used for the quantitative analysis of chiral amino acids.

Preferably, the lower detection limit of the amino acid solution is 0.38 μM-0.44 μM.

In the present invention, enzymes and fluorescent molecules are sequentially loaded into hierarchical porous metal-organic frameworks by means of post-fixation. The strong exoskeleton of metal-organic framework materials protects the protein structure of enzymes, improves the stability of enzymes, and its hierarchical porous structure will also The fluorescent molecules are separated and fixed on the metal nodes, which avoids the aggregation and quenching of fluorescent molecules; its large pore size and high porosity are conducive to the rapid diffusion and reaction of substances, and the cascade reaction is used in the detection process, that is, L-amino acid oxidation The oxidative deamination reaction between the enzyme and L-amino acid generates H ₂ O ₂ , and then H ₂ O ₂ undergoes a ring-opening reaction with the fluorescent molecule PF to generate strong fluorescence, which is detected by a fluorescence spectrophotometer to achieve the chiral detection of amino acids. Purpose. The preparation method according to the present invention has a high degree of extensibility, and realizes chiral recognition and quantitative analysis of different amino acids by loading different natural amino acid oxidases, providing a simple and efficient method for designing enantioselective and chemoselective fluorescent sensors. An effective approach is of great significance for the rapid and accurate chiral recognition and quantitative analysis of amino acids. The invention fixes different amino acid oxidases as the chiral recognition center, and can identify and analyze the chiral amino acid with high sensitivity and high specificity. The method has the advantages of convenient synthesis, mild preparation conditions, convenient instrument operation, low technical requirements, high analytical sensitivity, strong specificity and excellent anti-interference ability. The hierarchical porous metal-organic framework chiral sensing probe of the present invention can detect 8 kinds of amino acids in HEPES buffer solution, namely phenylalanine, leucine, methionine, tryptophan, histidine, isoleucine, The L-enantiomer of tyrosine and cysteine can be specifically and selectively recognized, and the L-amino acid can be linearly determined in the low concentration range below 100μM, and has a low detection limit (0.38μM-0.44μM), High sensitivity, strong specificity, and excellent anti-interference ability; and the detection instrument is simple, easy to operate, and low in technical requirements. The strategy provided by the present invention has universal applicability, simply replacing L-amino acid oxidase with L-glutamic acid oxidase can realize the selective recognition and content analysis of free L-glutamic acid in the aqueous phase. And it also has high specificity, low detection limit, and strong anti-interference ability.

Description of drawings

Fig. 1 is the process flow diagram of the preparation method of the hierarchical porous metal organic framework chiral sensing probe according to the present invention;

Fig. 2 is the proton nuclear magnetic resonance spectrum figure of the fluorescent molecule (PF) of hierarchical porous metal organic framework chiral sensing probe according to the present invention;

Fig. 3 is the adsorption kinetic curve of the hierarchical porous metal organic framework chiral sensing probe according to the present invention when loading L-amino acid oxidase L-AAO and fluorescent molecule PF;

Fig. 4 is the XRD spectrum of the hierarchical porous metal organic framework chiral sensing probe according to the present invention;

5 is a scanning electron microscope image of a hierarchical porous metal organic framework chiral sensing probe according to the present invention;

Fig. 6 is a nitrogen adsorption diagram and a pore size distribution diagram of the hierarchical porous metal organic framework chiral sensing probe according to the present invention;

Fig. 7 is the two enantiomeric kinetic response spectra of the hierarchical porous metal organic framework chiral sensing probe to phenylalanine according to the present invention;

Fig. 8 is the two enantiomeric kinetic response spectra of the hierarchical porous metal organic framework chiral sensing probe to leucine according to the present invention;

Fig. 9 is the two enantiomeric kinetic response spectra of the hierarchical porous metal-organic framework chiral sensing probe to methionine according to the present invention;

Fig. 10 is the two enantiomeric kinetic response spectra of the hierarchical porous metal organic framework chiral sensing probe to tryptophan according to the present invention;

Fig. 11 is the two enantiomeric kinetic response spectra of the hierarchical porous metal organic framework chiral sensing probe to histidine according to the present invention;

Fig. 12 is the two enantiomeric kinetic response spectra of the hierarchical porous metal organic framework chiral sensing probe to isoleucine according to the present invention;

Fig. 13 is the two enantiomeric kinetic response spectra of tyrosine according to the hierarchical porous metal organic framework chiral sensing probe of the present invention;

Fig. 14 is the two enantiomeric kinetic response spectra of cysteine according to the hierarchical porous metal organic framework chiral sensing probe of the present invention;

Fig. 15 is the fluorescence titration spectrum of the L-enantiomer of phenylalanine according to the hierarchical porous metal organic framework chiral sensing probe of the present invention;

Fig. 16 is the fluorescence titration spectrum of the L-enantiomer of leucine according to the hierarchical porous metal organic framework chiral sensing probe of the present invention;

Fig. 17 is the fluorescence titration spectrum of the L-enantiomer of methionine according to the hierarchical porous metal organic framework chiral sensing probe of the present invention;

Fig. 18 is a fluorescence titration spectrum of the L-enantiomer of tryptophan by the hierarchical porous metal organic framework chiral sensing probe according to the present invention;

Fig. 19 is the fluorescence titration spectrum of the L-enantiomer of histidine for the hierarchical porous metal organic framework chiral sensing probe according to the present invention;

Fig. 20 is the fluorescence titration spectrum of the L-enantiomer of isoleucine according to the hierarchical porous metal organic framework chiral sensing probe of the present invention;

Fig. 21 is the fluorescence titration spectrum of the L-enantiomer of tyrosine according to the hierarchical porous metal organic framework chiral sensing probe according to the present invention;

Fig. 22 is the fluorescence titration spectrum of the L-enantiomer of cysteine according to the hierarchical porous metal organic framework chiral sensing probe according to the present invention;

Fig. 23 is an image of the anti-interference and selective detection results of the hierarchical porous metal organic framework chiral sensing probe according to the present invention;

Fig. 24 is the fluorescent kinetic curve of the hierarchical porous metal organic framework chiral sensing probe according to the present invention in response to two enantiomers of glutamic acid, the fluorescence titration spectrum of L-glutamic acid, and the response of L-glutamic acid An image of the selective detection of glutamate.

detailed description

Below in conjunction with the drawings, preferred embodiments of the present invention are given and described in detail.

As shown in Figure 1, the preparation method of the hierarchical porous metal-organic framework chiral sensing probe according to the present invention includes the steps of: providing a MOFs precursor solution; generating a hierarchical porous UiO-66 precursor (HPUiO-66-as ); obtain hierarchical porous UiO-66 nanoparticles (HPUiO-66) by activating and removing templates; immobilize L-amino acid oxidase (L-AAO) on hierarchical porous UiO-66 nanoparticles (HPUiO-66), and obtain immobilized Hierarchical porous metal-organic framework for enzymes (L-AAO@HPUiO-66), in which L-amino acid oxidase (L-AAO) is loaded in the pores of hierarchically porous UiO-66 nanoparticles (HPUiO-66) by adsorption; Molecules (PF) were immobilized on the hierarchical porous metal-organic framework (L-AAO@HPUiO-66) immobilized with enzymes to obtain a hierarchical porous metal-organic framework chiral sensing probe (L-AAO&PF@HPUiO-66), in which, Fluorescent molecules (PF) are loaded into the pores of hierarchically porous UiO-66 nanoparticles (HPUiO-66) by adsorption.

Example 1

1.1 Synthesis of fluorescent molecules (PF)

3.15 g of 1,2,4-benzenetricarboxylic acid, 5.19 g of 3-bromophenol and 15 mL of methanesulfonic acid were added to the three-necked flask. And heated under reflux at 135° C. for 72 hours, after natural cooling, the mixture was added into 120 mL ice deionized water, stirred to obtain a green solid, and then the obtained solid was collected by vacuum filtration. Wash several times with a mixed reagent of pyridine and acetic anhydride (v/v=1:3), and recrystallize three times at 100°C with a mixed reagent of acetic anhydride and pyridine (v/v=2:1), and then use the concentration Wash with 1 mol L ^-1 hydrochloric acid for several times, and finally, vacuum-dry at 85°C for 12 hours to obtain a white solid powder.

The bis(pinacol) diboron with a quality of 400mg, the above-mentioned solid powder with a quality of 200mg, the Pd(dppf)Cl ₂ with a quality of 98.9mg, and the anhydrous potassium acetate with a quality of 510mg are loaded into a dry three-neck The flask was evacuated and then backfilled with nitrogen. This process is repeated at least three times. Then a volume of 5 mL of dry, degassed N,N-dimethylformamide was added. The mixed solution was then stirred at room temperature for 5 minutes and then heated at reflux at 85 °C for 3 h. After the reaction was cooled naturally, the solvent was removed by rotary evaporation, and the silica gel chromatography column equilibrated with dichloromethane was used to carry out gradient washing with a mixed solvent of dichloromethane and methanol to purify the crude product, and then a minimum amount of anhydrous ether was added dropwise to A light brown solid was precipitated, washed several times with anhydrous ether, and finally dried in vacuo at 40° C. for 12 h to obtain a bone white powder (PF).

The H NMR spectrum of Figure 2 proves the successful synthesis of PF.

1.2 Synthesis of Hierarchically Porous UiO-66 Nanoparticles

At room temperature, 150 mg of F127 was dissolved in 9 mL of deionized water, and then 750 mg of NaClO ₄ ·H ₂ O and 450 μL of acetic acid were ultrasonically dispersed in the F127 solution. Subsequently, 249 mg of terephthalic acid and 274 mg of ceric ammonium nitrate were stirred and dispersed in the above solution. Then, the mixed liquid was stirred and reacted at 60° C. for 20 minutes. The mixture was centrifuged to give a solid product. Then wash the material with deionized water, N,N-dimethylformamide, and absolute ethanol, and soak the material in absolute ethanol at 60°C for 48 hours. During this period, the ethanol is constantly replaced to remove all F127 in the pores of the material , after washing and drying, a hierarchical porous metal-organic framework material (HPUiO-66) with a size of 700nm-800nm was finally obtained.

1.3 Immobilization of L-amino acid oxidase (L-AAO) to obtain a hierarchical porous metal-organic framework (L-AAO@ HPUiO-66)

Dissolve 2.5 mg of L-amino acid oxidase in 2.5 mL of deionized water, and then disperse 2.5 mg of HPUiO-66 nanoparticles in the above enzyme solution. and stirred at 25°C for 4 hours. The enzyme-immobilized HPUiO-66 material (L-AAO@HPUiO-66) was collected by centrifugation and washed several times with deionized water to remove the enzyme adsorbed on the surface of HPUiO-66.

1.4 Immobilizing fluorescent molecules (PF) to obtain hierarchical porous metal-organic framework chiral sensing probes (L-AAO&PF@ HPUiO-66)

The fluorescent molecule PF with a mass of 5 mg was dissolved in 5 mL of deionized water, and then 2.5 mg of L-AAO@HPUiO-66 nanoparticles were dispersed in the above solution. And stirred at 25°C for 30 min in the dark. The HPUiO-66 materials immobilized with enzymes and fluorescent molecules (L-AAO&PF@HPUiO-66) were collected by centrifugation and washed several times with deionized water to remove the PF adsorbed on the surface.

Fig. 3 is the adsorption kinetics diagram of L-amino acid oxidase L-AAO and fluorescent molecule PF. Figure 4 is the XRD pattern of HPUiO-66 and L-AAO&PF@HPUiO-66. It can be seen that whether it is the hierarchical porous metal organic framework material HPUiO-66 or the hierarchical porous metal organic framework chiral probe material L-AAO&PF@HPUiO- 66, both maintain the characteristic peaks and high crystallinity of UiO-66 crystal; Figure 5 is the SEM image of HPUiO-66(A) and L-AAO&PF@HPUiO-66(B), which shows that HPUiO-66 and L-AAO&PF @HPUiO-66 has a regular pore structure with a particle size between 700nm and 800nm. Figure 6 shows the nitrogen adsorption-desorption curves and pore size distribution of HPUiO-66 and L-AAO&PF@HPUiO-66. It can be seen that the specific surface area, porosity, and pore diameter of L-AAO&PF@HPUiO-66 are greatly reduced compared with HPUiO-66. It was proved that L-AAO and PF were loaded into the pores of HPUiO-66 instead of being adsorbed on the surface. The pore structure parameters of HPUiO-66 and L-AAO&PF@HPUiO-66 are given in Table 1 below.

Table 1

sample Aperture (nm) Pore volume (cm³/g) Specific surface area (m²/g) HPUiO-66 9.2 0.626 1217.3 L-AAO&PF@HPUiO-66 6.7 0.142 262.0

1.5 Hierarchical porous metal-organic framework chiral sensing probe (L-AAO&PF@HPUiO-66) for chirality of amino acids identify

Disperse 5 mg of L-AAO&PF@HPUiO-66 probe material in 50 mL of HEPES buffer (20 mM, pH 7.4) to form a 100 mg L ^-1 probe solution, and 100 μL of L/D-amino acid solution (2 mM L ^-1 ) Add to 1.9 mL of the above probe solution, and incubate the reaction at 37° C. under dark conditions. Then, the fluorescence intensity of the mixture at λ=520 nm was detected at fixed time intervals. From the images in Figure 7-Figure 14, it can be seen that the L-AAO&PF@HPUiO-66 probe is sensitive to 8 kinds of amino acids (phenylalanine, leucine, methionine, tryptophan, histidine, isoleucine, tyrosine acid, cysteine) have chiral sensing responses. An increase in fluorescence was shown for the L-enantiomer, whereas there was no response for the corresponding D-enantiomer.

1.6 Hierarchical porous metal-organic framework chiral sensing probe (L-AAO&PF@HPUiO-66) for amino acid content analyze

Disperse 5 mg of L-AAO&PF@HPUiO-66 probe material in 50 mL of HEPES buffer (20 mM, pH 7.4) to form a 100 mg L ^-1 probe solution, and add 100 μL of L-amino acid solutions of different concentrations to 1.9 In mL of the above probe solution, the reaction was incubated at 37° C. in the dark until equilibrium, and the fluorescence intensity of the above reaction mixture at λ=520 nm was measured by a fluorescence spectrometer. From the images in Figure 15-Figure 22, it can be seen that when the L-enantiomers of the above 8 kinds of amino acids are added, the fluorescence intensity of the probe solution increases regularly with the increase of the concentration of L-amino acids. Within the concentration range, the probe can perform linear analysis on L-amino acids, the correlation coefficient is as high as 0.99, and it has a lower detection limit LOD (0.38μM-0.44μM). Table 2 below provides a summary of the fluorescence detection characteristics of the probes for the above 8 L-amino acids.

Table 2

1.7 Hierarchical porous metal-organic framework chiral sensing probe (L-AAO&PF@HPUiO-66) in interfering ions and chemical Fluorescent sensing and detection of amino acids in the presence of complexes

Interfering substances (Na ⁺ , K ⁺ , Mg ²⁺ , Al ³⁺ , Br ^- , Cl ^- , HCO ₃ ^- , NO ₂ ^- , glucose (GLU), glutathione (GSH), cholesterol (CHOL)) Dissolved in the probe solution at a concentration of 100 μM, and the probe solution was subjected to fluorescence measurement before and after adding 100 μL of L-phenylalanine (100 μM). The fluorescence intensity at λ=520nm was obtained. A in Figure 23 shows that the influence of the above-mentioned interfering substances on the fluorescence characteristics of the probe itself is negligible, and B shows that when the interfering substances exist in the mixed solution to be detected, they have no effect on the detection results of L-amino acids.

1.8 Hierarchical porous metal-organic framework chiral sensing probe (L-AAO&PF@HPUiO-66) in a simulated physiological environment Fluorescent sensing and detection of amino acids in the environment

18，

)与人体血浆浓度相一致，在37℃下用三-(羟甲基)氨基甲烷[(CH₂OH)₃CNH₂]和盐酸(HCl)将液体的缓冲pH值调节为7.4。并按照1.6分 级多孔金属有机骨架手性传感探针(L-AAO&PF@HPUiO-66)对氨基酸的含量分析的测试过程，在不同浓度的L-苯丙氨酸和D-苯丙氨酸混合溶液中进行L-苯丙氨酸的选择性传感，并同时进行四组平行实验，得到检测平均浓度。下表3的结果验证了L-AAO&PF@HPUiO-66探针在模拟生理条件下对L-苯丙氨酸的传感能力。Under simulated physiological conditions, L-phenylalanine, D-phenylalanine and L-AAO&PF@HPUiO-66 probe materials are dissolved in simulated body fluid (SBF), wherein the SBF solution configuration method is: NaCl , NaHCO ₃ , KCl, K ₂ HPO ₄ , MgCl ₂ , CaCl ₂ , Na ₂ SO ₄ dissolved in deionized water, inorganic ion concentration (mM: Na ⁺ 142, K ⁺ 5, Mg ²⁺ 1.5, Ca ²⁺ 2.5 , Cl ^- 149,

18,

) consistent with human plasma concentrations, the buffered pH of the liquid was adjusted to 7.4 with tris-(hydroxymethyl)aminomethane [(CH ₂ OH) ₃ CNH ₂ ] and hydrochloric acid (HCl) at 37°C. And according to the test process of 1.6 hierarchical porous metal organic framework chiral sensing probe (L-AAO&PF@HPUiO-66) for the content analysis of amino acids, in different concentrations of L-phenylalanine and D-phenylalanine The selective sensing of L-phenylalanine was carried out in the mixed solution, and four groups of parallel experiments were carried out at the same time to obtain the average concentration of detection. The results in Table 3 below verify the sensing ability of the L-AAO&PF@HPUiO-66 probe to L-phenylalanine under simulated physiological conditions.

table 3

sample Concentration added (μM) Determination concentration (μM)±σ Recovery rate(%) L-Phe/D-Phe 5/100 5.2±0.3 104±5 L-Phe/D-Phe 20/100 19.6±0.6 98±3 L-Phe/D-Phe 40/100 41.6±0.5 104±1 L-Phe/D-Phe 60/100 60.4±1.0 101±2

Example 2

Referring to 1.2-1.4 of Example 1, the immobilized amino acid oxidase was changed, and L-glutamate oxidase (L-GOX) was immobilized in hierarchical mesoporous metal-organic framework microspheres to obtain L-GOX&PF@HPUiO-66 probe needle material.

Referring to 1.5 of Example 1, change the L/D-amino acid to L/D-glutamic acid. It can be seen from Figure 24A that the L-GOX&PF@HPUiO-66 probe has chiral sensing ability for L-glutamic acid.

Referring to 1.6 of Example 1, change the L-AAO&PF@HPUiO-66 probe material to L-GOX&PF@HPUiO-66 probe material; change the L-amino acid to L-glutamic acid. It can be seen from Figure 24B and Figure 24C that the L-GOX&PF@HPUiO-66 probe can linearly analyze L-glutamic acid within 0-100 μM.

Add 100 μL of 19 amino acid solutions (100 μM) with chiral enantiomers to 1.9 mL of L-GOX&PF@HPUiO-66 probe solution (100 mg L ^-1 ). Incubate at 37° C. in the dark until the reaction is balanced, and measure the fluorescence intensity of the above reaction mixture at λ=520 nm by using a fluorescence spectrometer. Figure 24D shows that the probe has high selectivity to L-glutamic acid, and has no response to other L-amino acids and D-amino acids.

See 1.8 of Example 1, change L-phenylalanine and D-phenylalanine to L-phenylalanine, L-leucine, L-methionine, L-tryptophan, L-histidine , L-isoleucine, L-tyrosine, L-cysteine and D-glutamic acid. The results in Table 4 below demonstrate the sensing ability of the L-GOX&PF@HPUiO-66 probe to L-glutamic acid under simulated physiological conditions.

Table 4

的Hofmeister盐化阴离子作用，合成出分级多孔金属有机骨架材料。并通过后吸附的方式，将不同类型的氨基酸氧化酶以及荧光分子装载进入分级多孔金属有机骨架材料的孔道中，从而实现氨基酸的手性识别和含量分析。此方法具有普遍适用性，为生物分子荧光传感提供了新的方法和思路。The present invention adopts block copolymer F127 template and

The Hofmeister salinization anion, synthesized hierarchical porous metal-organic framework materials. And through the post-adsorption method, different types of amino acid oxidases and fluorescent molecules are loaded into the pores of the hierarchical porous metal organic framework material, so as to realize the chiral recognition and content analysis of amino acids. This method has universal applicability and provides a new method and idea for biomolecular fluorescence sensing.

What is described above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Various changes can also be made to the above embodiments of the present invention. That is to say, all simple and equivalent changes and modifications made according to the claims and description of the application for the present invention fall within the protection scope of the claims of the patent of the present invention. What is not described in detail in the present invention is conventional technical content.

Claims (9)

1. A preparation method for a hierarchical porous metal organic framework chiral sensing probe, characterized in that the preparation method comprises the steps: S1, providing hierarchical porous metal-organic framework microspheres, which have a regular pore structure; S2, immobilizing amino acid oxidase on hierarchical porous metal-organic framework microspheres to obtain hierarchical porous metal-organic framework microspheres immobilized with enzymes, wherein the amino acid oxidase is selected from L-amino acid oxidase and L-glutamic acid oxidase and at least one of the group consisting of L-tryptophan oxidase, the amino acid oxidase is loaded in the pores of the hierarchical porous metal-organic framework microspheres by adsorption; S3, immobilizing fluorescent molecules on hierarchical porous metal-organic framework microspheres immobilized with enzymes to obtain hierarchical porous metal-organic framework chiral sensing probes, wherein the fluorescent molecule is 3-Oxo-3', 6'-bis( 4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-spiro[isobenzofuran-1,9'-tonne]-6-carboxylic acid , fluorescent molecules are loaded in the pores of hierarchical porous metal-organic framework microspheres by adsorption. 2. preparation method according to claim 1, is characterized in that, described step S1 comprises: S11, using the block copolymer PEO ₁₀₆ PPO ₇₀ PEO ₁₀₆ to provide a metal organic framework precursor solution for the template agent; S12, generation of hierarchical porous metal-organic framework precursors by solvothermal; S13, Hierarchical porous metal-organic framework microspheres with large pore size obtained by activation and detemplating. 3. A hierarchical porous metal-organic framework chiral sensing probe obtained by the preparation method according to claim 1 or 2, characterized in that amino acid oxidase and fluorescent molecules are coupled on the surface of the hierarchical porous metal-organic framework microspheres on the carrier. 4. The application of the hierarchical porous metal organic framework chiral sensing probe according to claim 3, characterized in that the hierarchical porous metal organic framework chiral sensing probe has a chiral sensing response to amino acids. 5. The application according to claim 4, characterized in that the amino acid is phenylalanine, leucine, methionine, tryptophan, histidine, isoleucine, tyrosine, and/or semi cystine. 6. The application according to claim 4, characterized in that the hierarchical porous metal-organic framework chiral sensing probe shows enhanced fluorescence to the L-enantiomer of amino acids, but has no response to the D-enantiomer of amino acids. 7. The application according to claim 4, characterized in that the hierarchical porous metal organic framework chiral sensing probe is dispersed in the HEPES buffer to form a probe solution, the amino acid solution is added to the probe solution for incubation, and the fluorescence spectrometer is used to Measure its fluorescence intensity. 8. The application according to claim 7, characterized in that within the concentration range of the amino acid solution of 0-100 μM, the linear analysis of the amino acid is carried out by using a hierarchical porous metal-organic framework chiral sensing probe. 9. The application according to claim 7, characterized in that the lower detection limit of the amino acid solution is 0.38 μM~0.44 μM.

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